This application is in response to a Request for Applications for New Methodologies in Natural Product Chemistry (RFA-RM-09-005). We propose a campaign to establish a new program of natural product discovery based on a) a new biological source in the form of cave derived microorganisms and b) a new analytical method based on ion mobility mass spectrometry (IM-MS). These two aspects will open up a new reservoir of biodiversity for the discovery of natural products and provide a new method to accelerate the discovery of compounds in crude extracts and reduce the labor involved in their isolation. The labor and time savings derive from the ability to prioritize metabolites for isolation and structure elucidation based on their IM-MS measured properties. Aim 1. Develop natural product analysis using ion mobility mass spectroscopy (IM-MS). IM-MS is a form of 2-dimensional mass spectrometry that plots both mass/charge ratio and gas-phase collision cross- sectional area, which may be directly related to molecular conformation and functionality. We will evaluate the correlation of mass-collision area ratio to natural product class and compare these correlations to computationally predicted values. These data will map compound structure, class and conformation to IM-MS values. Aim 2. Develop cave microorganisms as a new source of natural product diversity. The isolation, cryogenic preservation and genotaxonomic characterization of culturable hypogean myxobacteria and actinobacteria will provide a new reservoir for natural product discovery and a source of extract material for the subsequent aims. Aim 3. Develop IM-MS as a method to identify candidates in extracts. A subset of taxonomically unique organisms will be evaluated for secondary metabolic potential by IM-MS. Compounds with IM-MS properties that distinguish compound class, functionality or unique conformation will be targeted for isolation/structure elucidation. The aims are parallel, but synergistic. New methods for identifying secondary metabolites from complex sources are essential for accelerating discovery. New sources of biodiversity are continuously needed to expand access to their biosynthetic molecular diversity. Cave microorganisms will provide a biological source of extracts needed to develop IM-MS as tool for identification of secondary metabolites in extracts. The natural products discovered by proposed methodologies will be available to the NIH Molecular Libraries Program (MLP) including Molecular Libraries Probe Production Centers Network (MLPCN), Molecular Libraries Small- Molecule Repository (MLSMR) and PubChem. PUBLIC HEALTH RELEVANCE: Natural products are an excellent source for drugs, but they are difficult to discover as they must be identified as minor components of complex extracts from biological sources. We propose to develop two-dimensional separations on the basis of molecular structure and mass to guide the identification of natural products from complex biological sources. Furthermore, cave derived microorganisms, a previously untapped source of biodiversity, will be investigated for natural product discovery.